Clostridium perftingens enterotoxin (CPE) causes the gastrointestinal (GI) symptoms ofthe 2nd most common bacterial foodborne disease in the USA and 5-15% of antibiotic-associated diarrhea cases. To prevent/control CPE-associated GI disease, and further development ofCPE as an anti-cancer agent, this project seeks to understand CPE's unique mechanism of action during GI disease. To progress towards this goal, the MERIT extension will. Aim A, further evaluate CPE interactions with claudin receptors by determining the structure of claudin-4 and CPE bound to claudin-4, map claudin-4 residues essential for CPE binding, evaluate if claudins with low CPE binding affinity can still convey CPE cytotoxicity and investigate the use of claudin receptor decoys as therapeutics against CPE-mediated GI disease; Aim B, continue analysis of post-binding steps in CPE action by mass spectrometry analysis of CPE complexes, evaluate the process of CPE complex formation using claudin- expresing transfectants, perform scanning transmission electron microscopy to evaluate complex mass and homogeneity, investigate the role of mitochondrial proteins in CPE-induced cell death, evaluate CPE complex formation in vivo, and determine if CPE induces intestinal inflammatory responses that might contribute to GI disease; Aim C, conduct additional study of CPE structure/function analyses by finishing the structure of CPE, perform site-directed mutagenesis to confirm key functional regions suggested by analysis ofthe CPE structure, evaluate the contribution of a putative CPE membrane-spanning domain to CPE action by mutagenesis and biophysical approaches (fluorescence spectroscopy and SCAM) and test whether CPE amino acids 45-52 represent an oligomerization latch domain; Aim D, further analyze the molecular pathogenesis of CPE-positive type A isolates by testing if the cpe plasmid conjugatively transfers to normal flora C. perfringens strains in the mouse intestines; compare (by sequencing) the cpe locus organization in type A isolates vs. type C and D isolates; evaluate the molecular regulation of CPE expression by constructing SigF and SigG mutants; and test whether other toxins produced by CPE-positive type A isolates contribute to GI pathogenesis by constructing isogenic cpe, pfoA, pic and/or cpb2 knockout mutants and testing their virulence in rabbit ileal loops. RELEVANCE (See Instructions): The purpose of this project is to determine the action of Clostridium perfringens enterotoxin (CPE), which is responsible for the symptoms of the 2nd most common foodborne illness in the USA and many cases of antibiotic-associated diarrhea. These insights will generate specific therapeutics to prevent these illnesses and allow further development of CPE as a potential cancer therapeutic.